Figure 4.

SLC7A5 is a direct YAP/TAZ TEAD target gene to promote leucine uptake. (A) SLC7A5 expression is dependent on YAP/TAZ. qPCR of SLC7A5 mRNA levels in 293A WT cells compared to Y/T dbKO cells. (B) LATS1/2 dbKO increases SLC7A5 expression. qPCR of SLC7A5 mRNA levels from 293A WT cells compared to LATS1/2 dbKO cells. (C) The promoter of SLC7A5 contains several putative TEAD recognition motifs. Diagram depicting the upstream 2 kB of the SLC7A5 gene. TSS: transcription start site. ATG: start codon. Potential TEAD recognition motifs (CATTCC) are depicted with arrow heads. (D) YAP drives SLC7A5 promoter activity. The short (−1 075 to 0) or long (−2 000 to 0) fragment of SLC7A5 promoter region was fused to produce the luciferase reporters. These reporters were transfected into Y/T dbKO 293A cells in combination with YAP or vector control. Luciferase activity was measure and normalized to the co-transfected renilla control. Note that only the long form contains the predicted TEAD recognition motifs (upstream of −1 075). Data are means ± SD from triplicates in a representative experiment. (E) TEAD1 binds to the SLC7A5 promoter region. Chromatin immunoprecipitation (CHIP) with antibodies to endogenous TEAD1 (or control IgG) were carried out in 293A cells. The precipitated DNA was quantitated by real-time PCR analysis with primers specific for a promoter region (prom) or an intragenic control region (intra) of the indicated genes. CTGF (connective tissue growth factor) is a known direct target gene of the YAP-TEAD complex, and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) serves as an additional negative control. Data are means ± SEM of triplicates from a representative experiment. (F) Ectopic expression of active YAP or TAZ increases SLC7A5 mRNA expression. qPCR from Y/T dbKO cells expressing doxycycline-inducible active YAP/TAZ as depicted. The cells were serum starved and doxycycline-induced for 16 h. Data are means ± SEM of triplicates from a representative experiment. (G) Ectopic expression of active YAP or TAZ increases SLC7A5 protein levels. Experiments are similar to F. ^* denotes antibody (sc-101199) that recognizes both YAP and TAZ. (H) SLC7A5 expression is dependent on YAP-TEAD interaction. Western blots of cell lysates from Y/T dbKO cell lines stably re-expressing YAP, or the TEAD binding-deficient YAP mutant (S94A). Note that the TEAD binding-deficient S94A YAP does not rescue the expression of SLC7A5. (I) Leucine uptake is dependent on YAP-mediated TEAD activation. H³-leucine uptake assay was carried out on Y/T dbKO cell lines with stable expression of vector control, YAP, and the TEAD binding-deficient YAP S94A mutant. Error bars are means ± SEM of triplicates from a representative experiment.